Enzymatic method for preparing rebaudioside N

ABSTRACT

Provided is a method for preparing rebaudioside N using an enzymatic method, comprising using rebaudioside A or rebaudioside J as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of a UDP-glycosyl-transferase and/or a UDP-glycosyltransferase-containing recombinant cell to generate rebaudioside N.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing, (file name: 3711_9300000_SequenceListing.txt; size: 12,144 bytes; and date of creation: Apr. 15, 2019), filed herewith, is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to a method for preparing rebaudioside N using an enzymatic method, and in particular, relates to biological preparation method of rebaudioside N.

BACKGROUND

Sweeteners are a kind of food additives widely used in the production of foods such as beverages and confectionery. The sweeteners may be added either during the production of foods or used, through appropriate dilution, as substitutes for sucrose in household baking. The sweeteners include natural sweeteners and artificial sweeteners, the former including sucrose, high fructose corn syrup, honey, and the like, the latter including aspartame, saccharin, and the like. Stevioside is a type of natural sweetening agent extracted from Stevia rebaudiana plant, and now has been widely used in food and beverages. Extracts of Stevia rebaudiana contain a plurality of steviosides comprising rebaudioside, and naturally extracted steviosides have larger difference in composition batch by batch, which need to be subsequently purified.

The ratio of rebaudioside N in Stevia rebaudiana leaves is less than 1.5%. It is extremely difficult to obtain high-purity rebaudioside N by traditional extraction methods, which limits the in-depth study of rebaudioside N and hinders its commercial application.

SUMMARY

The technical problem to be solved by the present invention is to overcome the defects in the prior art, and provide a method for preparing rebaudioside N using an enzymatic method. According to the method, high-purity rebaudioside N products may be prepared with a low cost in a short production cycle.

The following technical solution is employed by the present invention to solve the technical problem described above.

A method for preparing rebaudioside N using an enzymatic method is provided. The method comprises: using rebaudioside A as a substrate, in the presence of a glycosyl donor, reacting under catalysis of a UDP-glycosyltransferase-containing recombinant cell and/or a prepared UDP-glycosyltransferase to generate the rebaudioside N.

A method for preparing rebaudioside N using an enzymatic method is further provided. The method comprises: using rebaudioside J as a substrate, in the presence of a glycosyl donor, reacting under catalysis of a UDP-glycosyltransferase-containing recombinant cell and/or, a prepared UDP-glycosyltransferase to generate the rebaudioside N.

Preferably, the glycosyl donor comprises one or two of a glucose-based donor and a rhamnosyl donor; wherein the glucose-based donor is UDP-glucose or a UDP-glucose regeneration system (2007, FEBS Letters, 581, 2562-2566) consisting of sucrose, sucrose synthase and UDP, and the rhamnosyl donor is UDP-rhamnose. The glucose-based donor is preferably the UDP-glucose regeneration system consisting of sucrose, sucrose synthase and UDP. The price of the UDP glucose is high. Therefore, use of the UDP-glucose regeneration system may greatly lower the cost.

Preferably, the UDP-glycosyltransferase (that is, uridine diphosphate glucanotransferase, which is referred to as UGT for short, and is known in the art) comprises one or two of UGT-A from Stevia rebaudiana and UGT-B from Oryza sativa.

Preferably, the UDP-glycosyltransferase comprises UGT-A from Stevia rebaudiana and UGT-B from Oryza sativa; the UDP-glycosyltransferase is added into the reaction system in two steps; wherein the UGT-B is added in the first step and the UGT-A is added in the second step. An amino acid sequence of the UGT-A has at least 60% identity to sequence 2 listed in a sequence listing; and/or an amino acid sequence of the UGT-B has at least 60% identity to sequence 4 listed in the sequence listing.

More preferably, the amino acid sequence of the UGT-A has at least 70% identity to sequence 2 listed in the sequence listing; and/or the amino acid sequence of the UGT-B has at least 70% identity to sequence 4 listed in the sequence listing.

Further, the amino acid sequence of the UGT-A has at least 80% identity to sequence 2 listed in the sequence listing; and/or the amino acid sequence of the UGT-B has at least 80% identity to sequence 4 listed in the sequence listing.

Still further, the amino acid sequence of the UGT-A has at least 90% identity to sequence 2 listed in the sequence listing; and/or the amino acid sequence of the UGT-B has at least 90% identity to sequence 4 listed in the sequence listing.

In some specific embodiments, the amino acid sequence of the UGT-A is absolutely identical to sequence 2 listed in the sequence listing; and/or the amino acid sequence of the UGT-B is absolutely identical to sequence 4 listed in the sequence listing.

Preferably, the UDP-glycosyltransferase is UGT-A from Stevia rebaudiana; wherein an amino acid sequence of the UGT-A has at least 60% identity to sequence 2 listed in a sequence listing.

More preferably, the amino acid sequence of the UGT-A has at least 70% identity to sequence 2 listed in the sequence listing.

Further, the amino acid sequence of the UGT-A has at least 80% identity to sequence 2 listed in the sequence listing.

Still further, the amino acid sequence of the UGT-A has at least 90% identity to sequence 2 listed in the sequence listing. In some specific embodiments, the amino acid sequence of the UGT-A is absolutely identical to sequence 2 listed in the sequence listing.

According to the present invention, the reaction is carried out in an aqueous phase system at a temperature of 4 to 50° C. and a pH value of 5.0 to 9.0. Preferably, the reaction is carried out in an aqueous phase system at a temperature of 35 to 45° C. and a pH value of 7.5 to 8.5.

More preferably, the reaction is carried out in a phosphate buffer solution.

More preferably, the reaction system comprises a UDP-glycosyltransferase recombinant cell and a cell permeabilizing agent. Further, cell permeabilizing agent is toluene; wherein the toluene has a volume specific concentration of 1 to 3% in the reaction system.

More preferably, all the starting materials used for the reaction are added into a reaction vessel, mixed uniformly and placed at a set temperature, and stirred for sufficient reaction. Upon completion of the reaction, a rebaudioside N product satisfying the using requirement may be obtained by purification. A specific purification method is post-treatment including resin separation. According to this purification method, a rebaudioside N product having a purity reaching 95% may be obtained.

Preferably, the recombinant cell is a microbial cell. More preferably, the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

According to a specific aspect of the present invention, a reaction substrate in the first step is rebaudioside A, and the UDP-glycosyltransferase is UGT-B from Oryza sativa, wherein an amino acid sequence of the UGT-B from Oryza sativa has at least 80% identity to sequence 4; and a reaction substrate in the second step is a reaction solution containing a reaction product rebaudioside J from the first step, and the UDP-glycosyltransferase is UGT-A from Stevia rebaudiana, wherein an amino acid sequence of the UGT-A from Stevia rebaudiana has at least 80% identity to sequence 2.

According to another specific aspect of the present invention, a reaction substrate is rebaudioside J, the UDP-glycosyltransferase is UGT-A from Stevia rebaudiana, wherein an amino acid sequence of the UGT-A from Stevia rebaudiana has at least 80% identity to sequence 2.

Compared with the prior art, the present invention has the following advantages by performing the foregoing technical solution:

The method for preparing rebaudioside N using an enzymatic method according to the present invention has a very significant application value. Since the microorganisms grow far more faster than the plants, by this method, the production cost may be greatly lowered, the production cycle may be shortened and the competitiveness of products may be greatly improved. In addition, the plants have a low content of stevioside, but contain a large number of steviosides with different structures. Therefore, it is difficult to extract purer products. As compared with the conventional technique of extracting rebaudioside N from the leaves of Stevia rebaudiana, the enzymatic synthesis method according to the present invention may achieve products having a higher purity, and inevitably promotes studies and application of novel stevioside-type rebaudioside N.

DETAILED DESCRIPTION OF THE INVENTION

Formula I and formula II are structural formulae of rebaudioside J and rebaudioside N.

The present invention mainly provides two routes for synthesizing the rebaudioside N.

Route One:

Route Two:

The UGT-A or UGT-B used in the present invention may be present in an enzyme lyophilized powder form or present in a recombinant cell.

The method for obtaining UGT-A or UGT-B is as follows:

Expression strains of recombinant Escherichia coli (or other microorganisms) are obtained by the molecular cloning technique and genetic engineering technique, and then the recombinant Escherichia coli is fermented to obtain recombinant cells containing the UGT-A or the UGT-B; or lyophilized powder of the UGT-A or the UGT-B is prepared from the recombinant cells.

The molecular cloning technique and the genetic engineering technique according to the present invention are both known. For details about the molecular cloning technique, reference may be made to Molecular Cloning: A laboratory Manual, third version (by Joseph Sambrook, 2005).

The expression steps of the recombinant strain herein constructed by employing genetic engineering technique are as follows:

(1) Desired gene fragments are genetically synthesized (based on sequence 1 and sequence 2 or sequence 3 and sequence 4 listed in the sequence listing), and ligated into a vector pUC57, wherein NdeI and BamHI enzyme digestion sites are loaded at two terminals thereof.

(2) By double enzyme digestion and linking, the gene fragments are inserted into the corresponding enzyme digestion sites of a expression vector pTE30a, such that the genes are placed under control of a T7 promoter.

(3) The recombinant plasmids are transformed and placed into the Escherichia coli BL21 (DE3), and a target protein expression is induced by IPTG, such that the recombinant Escherichia coli expression strains containing the UGT-A or the UGT-B are obtained.

The recombinant cells containing the UGT-A or the UGT-B, and the lyophilized powder of the UGT-A or the UGT-B are prepared by using the recombinant Escherichia coli expression strains containing the UGT-A or the UGT-B by the following steps:

The recombinant Escherichia coli expression strains containing the UGT-A or the UGT-B are inoculated at a proportion of 1% into a 4 ml liquid LB medium, and the medium is shaken (at a speed of 200 rpm) at 37° C. overnight; the overnight culture is transferred at an inoculation amount of 1% into a 50 ml liquid LB medium, and is inoculated by shaking (at a speed of 200 rpm) at 37° C. until the OD600 value reaches the range of 0.6 to 0.8; and then the IPTG having a concentration of 0.4 mM is added and overnight inoculation is carried out by shaking at 20° C. Upon completion of inducement, cells are centrifuged (8000 rpm and 10 min) and collected, the cells are resuspended by using a 5 ml 2 mmol/L phosphate buffer (having a pH value of 7.0) to obtain the recombinant cells. The obtained recombinant cells are further treated in ice bath and are ultrasonically crushed. A crushing liquid is centrifuged (8000 rpm and 10 min). Afterwards, a supernatant is collected and lyophilized for 24 h to obtain the lyophilized powder.

The present invention is described in detail hereinafter with reference to specific examples.

Example 1: Preparation of Recombinant Escherichia coli Cells Containing the UGT-A

In accordance with sequence 1 and sequence 2 listed in the sequence listing, the UGT-A gene fragments are genetically synthesized, NdeI and BamHI enzyme digestion sites are loaded at two terminals thereof respectively, and the gene fragments are linked to a vector pUC57 (produced by Suzhou GENEWIZ Biotechnology Co., Ltd). The UGT gene fragments were enzyme digested by using the restrictive endonucleases NdeI and BamHI, and purified fragments were recovered. The fragments were linked to the corresponding enzyme digestion site of the vector pTE30a by using a T4 ligase, and thus the fragments were transformed to the BL21 (DE3) strains.

The UGT strains were inoculated at a proportion of 1% into a 4 ml liquid LB medium, and the medium was shaken (at a speed of 200 rpm) at 37° C. overnight; the overnight culture was transferred at an inoculation amount of 1% into a 50 ml liquid LB medium, and was inoculated by shaking (at a speed of 200 rpm) at 37° C. until the OD600 value reached the range of 0.6 to 0.8; and then the IPTG having a concentration of 0.4 mM was added and overnight inoculation was carried out by shaking at 20° C. Upon completion of inducement, cells were centrifuged (8000 rpm and 10 min) and collected, the cells were resuspended by using a 5 ml 2 mmol/L phosphate buffer (having a pH value of 7.0) to obtain the recombinant cells containing the UGT-A.

Example 2: Preparation of UGT-A Lyophilized Powder

The recombinant cells containing the UGT-A prepared in Example 1 were further treated in ice bath and were ultrasonically crushed. A crushing liquid was centrifuged (8000 rpm and 10 min). Afterwards, a supernatant was collected and lyophilized for 24 h to obtain the lyophilized powder of the UGT-A.

Example 3: Preparation of Recombinant Escherichia coli Cells Containing the UGT-B

In accordance with sequence 3 and sequence 4, the UGT-B gene fragments were genetically synthesized, NdeI and BamHI enzyme digestion sites were loaded at two terminals thereof respectively, and the gene fragments were linked to a vector pUC57 (produced by Suzhou GENEWIZ Biotechnology Co., Ltd). The UGT gene fragments were enzyme digested by using the restrictive endonucleases NdeI and BamHI, and purified fragments were recovered. The fragments were linked to the corresponding enzyme digestion site of the vector pTE30a by using a T4 ligase, and thus the fragments were transformed to the BL21 (DE3) strains.

The UGT strains were inoculated at a proportion of 1% into a 4 ml liquid LB medium, and the medium was shaken (at a speed of 200 rpm) at 37° C. overnight; the overnight culture was transferred at an inoculation amount of 1% into a 50 ml liquid LB medium, and was inoculated by shaking (at a speed of 200 rpm) at 37° C. until the OD600 value reached the range of 0.6 to 0.8; and then the IPTG having a concentration of 0.4 mM was added and overnight inoculation was carried out by shaking at 20° C. Upon completion of inducement, cells were centrifuged (8000 rpm and 10 min) and collected, the cells were resuspended by using a 5 ml 2 mmol/L phosphate buffer (having a pH value of 7.0) to obtain the recombinant cells containing the UGT-B for catalysis.

Example 4: Preparation of UGT-B Lyophilized Powder

The recombinant cells containing the UGT-B prepared in Example 3 were further treated in ice bath and were ultrasonically crushed. A crushing liquid was centrifuged (8000 rpm and 10 min). Afterwards, a supernatant was collected and lyophilized for 24 h to obtain the lyophilized powder of the UGT-B.

Example 5: Synthesis of Rebaudioside N by Using Rebaudioside J as a Reaction Substrate Under Catalysis of UDP-Glycosyltransferase (Route One)

In this example, the rebaudioside N was catalytically synthesized by using the UGT-A lyophilized powder prepared by the method in Example 2. In this example, a UDP-glucose regeneration system consisting of sucrose, sucrose synthase from Arabidopsis thaliana (hereinafter referred to as AtSUS1) and UDP was used as a glucose-based donor.

1 L of 0.05 mol/L phosphate buffer (having a pH value of 8.0), 0.5 g of UDP, 1 g of rebaudioside J, 5 g of sucrose, 10 g of UGT-A lyophilized powder, and 0.5 g of AtSUS1 lyophilized powder were added sequentially into the reaction system and uniformly mixed, and the resulted solution was then placed in water bath at 40° C., stirred at a speed of 300 rpm, and reacted for 24 h. Upon completion of the reaction, 500 μl of the reaction solution was taken and added into an equivalent volume of non-aqueous methanol for uniform mixture, and the resulted solution was centrifuged at a speed of 8000 rpm for 10 min. Afterwards, a supernatant was filtered by a filter membrane and tested by a high performance liquid chromatography (chromatographic conditions: chromatography column: Agilent eclipse SB-C18 4.6×150 mm; test wavelength: 210 nm; mobile phase: 0.1% solution of formic acid:acetonitrile=65%:35%; flow rate: 1.0 ml/min; and column temperature: 30° C.). A conversion rate of the rebaudioside J was over 90%. 0.61 g of the rebaudioside N was obtained through purification by silica gel-resin separation, crystallization and the like post-treatments, and the obtained rebaudioside N had a purity greater than 95%.

Example 6: Synthesis of Rebaudioside N by Using Rebaudioside a as a Reaction Substrate Under Catalysis of UDP-Glycosyltransferase (Route Two)

In this example, the rebaudioside N was catalytically synthesized by using the UGT-A lyophilized powder prepared by the method in Example 2 and the UGT-B lyophilized powder prepared by the method in Example 4.

Reaction in the first step: 1 L of 0.05 mol/L phosphate buffer (having a pH value of 8.0), 2 g of UDP rhamnosyl, 1 g of rebaudioside A, and 10 g of UGT-B lyophilized powder were sequentially added into the reaction system and uniformly mixed, and then the resulted solution was placed in water bath at 40° C., stirred at a speed of 300 rpm, and reacted for 24 h. Reaction in the second step: Upon completion of the reaction in the first step, the reaction solution was boiled for 10 min, the pH value of the reaction solution was regulated to 8.0, 0.5 g of UDP, 5 g of sucrose, 10 g of UGT-A lyophilized powder and 3 g of AtSUSI lyophilized powder were added and uniformly mixed, and the resulted solution was then placed in water bath at 40° C., stirred at a speed of 300 rpm, and reacted from 24 h. Upon completion of the reaction, 500 μl of the reaction solution was taken and added into an equivalent volume of non-aqueous methanol for uniform mixture, and the resulted solution was centrifuged at a speed of 8000 rpm for 10 min. Afterwards, a supernatant was filtered by a filter membrane and tested by a high performance liquid chromatography (chromatographic conditions: chromatography column: Agilent eclipse SB-C18 4.6×150 mm; test wavelength: 210 nm; mobile phase: 0.1% solution of formic acid:acetonitrile=65%:35%; flow rate: 1.0 ml/min; and column temperature: 30° C.). A conversion rate of the rebaudioside A was over 90%. 0.58 g of the rebaudioside N was obtained through purification by silica gel-resin separation, crystallization and the like post-treatments, and the obtained rebaudioside N had a purity greater than 95%.

Example 7: Synthesis of Rebaudioside N by Using Rebaudioside J as a Reaction Substrate Under Catalysis of the Recombinant Cells Containing the UDP-Glycosyltransferase

In this example, the rebaudioside N was catalytically synthesized by using the recombinant cells containing the UGT-A prepared by the method in Example 1.

1 L of 0.05 mol/L phosphate buffer (having a pH value of 8.0), 0.5 g of UDP, 1 g of rebaudioside J, 5 g of sucrose, 40 g of UGT-A whole cells, and 10 g of AtSUS1 whole cells were added sequentially into the reaction system and uniformly mixed, and the resulted solution was then placed in water bath at 40° C., stirred at a speed of 300 rpm, and reacted for 24 h. Upon completion of the reaction, 500 μl of the reaction solution was taken and added into an equivalent volume of non-aqueous methanol for uniform mixture, and the resulted solution was centrifuged at a speed of 8000 rpm for 10 min. Afterwards, a supernatant was filtered by a filter membrane and tested by a high performance liquid chromatography (chromatographic conditions: chromatography column: Agilent eclipse SB-C18 4.6×150 mm; test wavelength: 210 nm; mobile phase: 0.1% solution of formic acid:acetonitrile=65%:35%; flow rate: 1.0 ml/min; and column temperature: 30° C.). A conversion rate of the rebaudioside J was over 90%. 0.54 g of the rebaudioside N was obtained through purification by silica gel-resin separation, crystallization and the like post-treatments, and the obtained rebaudioside N had a purity greater than 95%.

Example 8: Synthesis of Rebaudioside N by Using Rebaudioside a as a Reaction Substrate Under Catalysis of the Recombinant Cells Containing the UDP-Glycosyltransferase

Reaction in the first step: 1 L of 0.05 mol/L phosphate buffer (having a pH value of 8.0), 2 g of UDP rhamnosyl, 1 g of rebaudioside A, and 40 g of UGT-B whole cells were sequentially added into the reaction system and uniformly mixed, then placed in water bath at 40° C., stirred at a speed of 300 rpm, and reacted for 24 h. Reaction in the second step: Upon completion of the reaction in the first step, the reaction solution was boiled for 10 min, the pH value of the reaction solution was regulated to 8.0, 0.5 g of UDP, 5 g of sucrose, 40 g of UGT-A whole cells, and 10 g of AtSUSI whole cells were added and uniformly mixed, and the resulted solution was then placed in water bath at 40° C., stirred at a speed of 300 rpm, and reacted from 24 h. Upon completion of the reaction, 500 μl of the reaction solution was taken and added into an equivalent volume of non-aqueous methanol for uniform mixture, and the resulted solution was centrifuged at a speed of 8000 rpm for 10 min. Afterwards, a supernatant was filtered by a filter membrane and tested by a high performance liquid chromatography (chromatographic conditions: chromatography column: Agilent eclipse SB-C18 4.6×150 mm; test wavelength: 210 nm; mobile phase: 0.1% solution of formic acid:acetonitrile=65%:35%; flow rate: 1.0 ml/min; and column temperature: 30° C.). A conversion rate of the rebaudioside A was over 90%. 0.53 g of the rebaudioside N was obtained through purification by silica gel-resin separation, crystallization and the like post-treatments, and the obtained rebaudioside N had a purity greater than 95%.

The above examples are merely intended to describe the technical concept and characteristics of the present invention, such that a person skilled in the art would better understand the disclosure of the present invention and practice the present invention. However, these examples shall not be construed to limit the protection scope of the present invention. Various equivalent variations or polishments made without departing from the spirit and essence of the present invention shall all be considered as falling within the protection scope of the present invention. 

The invention claimed is:
 1. An enzymatic method for preparing rebaudioside N, the method comprising reacting rebaudioside J with a glycosyl donor in a reaction system comprising: recombinant cells comprising a UDP-glycosyltransferase and/or a UDP-glycosyltransferase prepared therefrom, wherein the UDP-glycosyltransferase has the amino acid sequence according to SEQ ID NO:
 2. 2. The method of claim 1, wherein the glycosyl donor is UDP-glucose or a UDP-glucose regeneration system comprising sucrose, sucrose synthase and UDP.
 3. The method of claim 1, wherein the UDP-glycosyltransferase is UGT-A from Stevia rebaudiana.
 4. The method of claim 1, wherein the reaction is carried out in an aqueous system at a temperature of 35 to 45° C. and at a pH of 7.5 to 8.5.
 5. The method of claim 4, wherein the aqueous system comprises a phosphate buffer solution.
 6. The method of claim 4, wherein the aqueous system further comprises a cell permeabilizing agent.
 7. The method of claim 6, wherein the cell permeabilizing agent is toluene and wherein the toluene is present in the aqueous system at a concentration by volume of 1-3%.
 8. The method of claim 1, wherein the recombinant cell is a cell of a microorganism.
 9. The method of claim 8, wherein the microorganism is Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.
 10. The method of claim 1, further comprising purifying the rebaudioside N via resin isolation.
 11. The method of claim 10, wherein the rebaudioside N purified via resin isolation has a purity greater than 95%. 